Screening method of p21-CDK2 / Cyclin interface inhibitor

Abstract

The invention discloses a screening method of a p21-CDK2 / cyclin interface inhibitor. The screening method comprises the following steps: (1) constructing a protein interaction model containing p21-CDK2 / cyclin A; (2) calculating binding free energy of the model in the step (1) by using molecular dynamics software, and simulating and analyzing interacting amino acid sites; and 3) virtually screening out the small molecule compound inhibitor according to the simulated protein interaction amino acid region by using molecular docking software. The inhibitor can inhibit cell aging. The method can be used for screening drugs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for screening a p21-CDK2 / Cyclin interface inhibitor. Background Art

[0002] Cellular senescence is a stable and terminal state of growth arrest, in which cells cannot proliferate despite optimal growth conditions and mitogenic stimulation. Cellular senescence can be caused by a variety of factors, including ionizing radiation, oxidative stress, oncogene activation, etc. At present, the senescence mechanism is mainly completed through two pathways: Tumor Protein p53 (p53) / Cyclin Dependent Kinase Inhibitor 1A (p21) and / or Cyclin Dependent Kinase Inhibitor 2A (p16) / retinoblastoma protein (RB). After cells are irradiated, the p53 / p21 pathway is significantly activated, leading to cell cycle arrest and inducing cell senescence; the p16 / RB pathway is activated in the late stage of radiation damage to maintain cell senescence. Cyclin-dependent kinase (CDK) / cyclin complexes are key protein complexes that regulate the cell cycle and mitosis. p21 protein is a CDK inhibitory protein that plays a vital role in the regulation of the cell cycle. p21 protein can form complexes with a variety of CDK proteins, thereby inhibiting the activity of CDK and leading to cell cycle arrest. After radiation damage, the expression of p21 is upregulated, which in turn continuously inhibits the activity of protein complexes such as CDK2 / Cyclin A and CDK2 / Cyclin E, prompting cells to enter irreversible cell cycle arrest and aging. In recent years, as a marker of aging and tumor markers, the study of p21 and its related inhibitors has received increasing attention from scientific researchers. However, as a member of the intrinsically disordered protein family, p21 protein lacks a stable three-dimensional structure in its natural state, making it difficult to design inhibitors targeting p21 itself; and no interface inhibitors targeting the interaction between p21 and CDK2 / CyclinA and E series protein complexes have been reported. Therefore, developing interface inhibitors that can target the p21-CDK2 / Cyclin A interaction and block cell senescence caused by radiation damage activating the p53 / p21 signaling pathway by inhibiting the interaction between p21 and protein complexes such as CDK2 / Cyclin A and CDK2 / Cyclin E is of great significance for anti-radiation therapy and aging research. Summary of the invention

[0003] The purpose of the present invention is to provide a method for screening a p21-CDK2 / Cyclin interface inhibitor, wherein the inhibitor can inhibit cell senescence.

[0004] The method comprises the following steps:

[0005] 1) Construct a model of p21-CDK2 / Cyclin A protein interaction;

[0006] 2) Using molecular dynamics software to calculate the binding free energy of the model in step 1) and simulate and analyze the interacting amino acid sites;

[0007] 3) Use molecular docking software to virtually screen small molecule compound inhibitors based on the simulated protein-protein interaction amino acid regions; select the top 100 small molecule compounds with docking scores, and select 20 small molecule compounds based on their structural characteristics;

[0008] 4) The activity of the screened small molecule compounds was verified using a cell radiation-induced aging model.

[0009] Preferably, in the above screening method of the present invention, the cell senescence refers to the senescence of cells after radiation damage.

[0010] Preferably, in the above screening method of the present invention, step 1) further comprises using the Alphafold protein structure database and the PDB protein database to construct the p21 and CDK2 / Cyclin A protein structures respectively, using ZDOCK software to complete the simulated docking of the p21 and CDK2 / Cyclin A protein complex, and screening out the p21-CDK2 / Cyclin A protein interaction model that is most similar to the p27-CDK2 / Cyclin A protein complex structure.

[0011] Preferably, in the above-mentioned screening method of the present invention, step 2) further comprises using AMBER software to perform molecular dynamics simulation, data analysis, binding free energy calculation, and hotspot amino acid residue simulation analysis on the protein model obtained in step 1) to obtain key amino acid residue sites involved in protein interaction.

[0012] Preferably, in the above screening method of the present invention, step 2) further comprises using Autodock_Vina molecular docking software to virtually screen out small molecule compound inhibitors that inhibit protein-protein interactions based on key amino acid residue sites of protein interactions.

[0013] Preferably, in the screening method of the present invention, the verification in step 4) comprises:

[0014] a) Using gamma rays to irradiate bone marrow mesenchymal stem cells (BM-MSCs) to construct a cell radiation-induced aging model;

[0015] b) After the small molecule compound screened in step 3 is co-incubated with BM-MSC, the cells are irradiated with gamma rays, and the inhibitory activity of the small molecule compound on cell radiation-induced senescence is detected by β-galactosidase staining experiment and / or flow cytometry.

[0016] Specifically, the present invention uses the Alphafold protein structure database and the PDB protein database to construct the p21 and CDK2 / Cyclin A protein structures respectively, and then uses the ZDOCK software to complete the p21 and CDK2 / Cyclin A protein complex simulation docking to obtain multiple docking models, and based on the p27-CDK2 / Cyclin A crystal structure reported in the literature, the p21-CDK2 / Cyclin A protein interaction model that is most similar to the p27-CDK2 / Cyclin A protein complex structure is selected from the docking model, and then the protein model is subjected to molecular dynamics simulation, data analysis, binding free energy calculation, and hotspot amino acid residue simulation analysis using AMBER software to clarify the key amino acid residues involved in protein interaction. According to the amino acid sites in the key region of protein interaction, the Autodock_Vina molecular docking software is used to virtually screen out small molecule inhibitors that inhibit protein-protein interactions, and finally a small molecule inhibitor that effectively inhibits aging after cell radiation damage is screened out using cell experiments. At present, there is almost no report on the research on preventing healthy cells from aging by targeting the specific signaling pathways of cell aging. Therefore, the present invention is expected to provide a new strategy for anti-aging drug research. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 (a) is the p21-CDK2 / Cyclin A protein interaction binding model simulated by ZDOCK software, (b) is the simulation analysis diagram of the key amino acid residues of the p21 protein, (c) is the CDK2 protein, and (d) is the Cyclin A protein interaction;

[0018] Figure 2 Schematic diagram of docking of CDK2 / Cyclin A protein and the screened small molecule compounds;

[0019] Figure 3 is the survival rate of BM-MSCs proliferation experiment under different radiation doses;

[0020] Figure 4The senescence (a) and apoptosis (b) ratios of BM-MSCs at 1, 4, and 7 days after irradiation with 3 Gy, 6 Gy, 9 Gy, 12 Gy, and 15 Gy radiation doses; (c) is the β-galactosidase staining image in the senescence ratio experiment of (a); (d) is the flow cytometry image in the apoptosis ratio experiment of (b);

[0021] Figure 5 The effects of small molecule compounds on the activity (a) and senescence (b) of BM-MSCs on day 4 before and after 9Gy radiation; (c) is the β-galactosidase staining image in the senescence ratio experiment of (b);

[0022] Figure 6 Statistical graph (a) and flow cytometry graph (b) showing the effects of small molecule compounds on BM-MSCs apoptosis on day 4 before and after 9 Gy radiation. DETAILED DESCRIPTION

[0023] The following examples are representative and are used to further illustrate and understand the essence of the present invention, but are not intended to limit the scope of the present invention. Any simple modifications and adaptations made within the essence of the present invention fall within the scope of the present invention.

[0024] Example 1 Construction of a screening method for p21-CDK2 / Cyclin interface inhibitors

[0025] The screening method of the present invention can be specifically implemented by the following steps:

[0026] 1) Download the PDB file of p21 protein (AF-P38936-F1) from the Alphafold protein structure database; download the PDB file of p27 / CDK2 / Cyclin A protein complex (1JSU) from the RCSB protein database, and then extract the CDK2 / Cyclin A protein structure for use.

[0027] 2) Using the ZDOCK protein docking website, p21 and CDK2 / Cyclin A were simulated for docking, and the top ten protein-protein interaction complexes were obtained.

[0028] 3) From the protein complex obtained in the previous step, the p21-CDK2 / cyclin A structural model that is most similar to the p27-CDK2 / Cyclin A crystal structure reported in the literature (Nature 382, ​​325–331 (1996), the entire text is incorporated by reference) was selected, and then the protein model was subjected to molecular dynamics simulation, data analysis, binding free energy calculation, and hot spot amino acid residue simulation analysis using AMBER software to identify the key amino acid residues involved in protein interaction, calculate the binding free energy of the protein model, and determine the key amino acid sites of the interaction between p21 and CDK2 / Cyclin A proteins through the key amino acids that contribute more to the binding free energy.

[0029] 4) By analyzing the binding free energy, it was found that the key amino acids involved in protein-protein interaction are Val18, Tyr19, Lys34 and Arg36 of CDK2, Trp217, Gln254, Ile281 and Thr282 of Cyclin A, and Arg20, Leu21, Phe22, Trp49, Trp65 and Tyr77 of p21 (see Figure 1 ).

[0030] 5) In the crystal structure of the interaction between p27 and CDK2 / Cyclin A reported in the literature (Nature 382, ​​325–331, 1996, the entire text is incorporated by reference), the three amino acid regions of Leu32-Phe33-Gly34 (LFG) of the p27 protein are the key sites for the interaction between p27 and CDK2 / Cyclin A. p21 and p27 have a high degree of homology, and simulations have found that p21 also has the same LFG region, and the binding site with Cyclin A is in the key amino acid regions of Gln254 and Thr282.

[0031] 6) The amino acid regions of Gln254 and Thr282 in Cyclin A were selected as docking pockets for virtual screening of small molecule inhibitors. Using the AutoDOCK_vina molecular docking software, the amino acid regions of Gln254 and Thr282 in Cyclin A were selected as docking pockets, and a total of about 510,000 small molecules from the Enamine and LifeChemical commercial compound libraries were selected for virtual screening (see Figure 2 ). Based on the small molecule compounds and Gln 254 and Thr 282 The amino acid residue binding force was scored, and the top 100 small molecule compounds with the best docking scores were selected from the scoring results. According to the structural characteristics of the small molecule compounds, 20 small molecule compounds were selected (see Table 1 below), and then these compound samples were purchased from merchants for the next stage of cell experiments.

[0032] Table 1. Numbers and screening scores of 20 compounds*

[0033] Compound No. ProductID Screening and Scoring Compound No. ProductID Screening and Scoring No.1 Z56773617 -9.7 No.11 Z535577226 -9.3 No.2 Z28635399 -9.7 No.12 Z25736755 -9.3 No.3 Z2233592864 -9.5 No.13 F6609-1964 -9.7 No.4 Z356370764 -9.5 No.14 F2774-1705 -9.4 No.5 Z131704342 -9.4 No.15 F2865-0108 -9.3 No.6 Z271090688 -9.4 No.16 F2042-0164 -9.3 No.7 Z278198738 -9.3 No.17 F0922-0913 -9.3 No.8 Z240395752 -9.3 No.18 F6727-8363 -9.3 No.9 Z45503561 -9.3 No.19 F3222-3374 -9.3 No.10 Z212730924 -9.3 No.20 F1142-3225 -9.2

[0034] *The screening score is provided by the AutoDock Vina software, which uses the scoring function vina score to evaluate the binding ability of molecular docking. It calculates the interaction energy and binding free energy between molecules to get a score. The lower the score, the stronger the binding ability.

[0035] Example 2 Cell activity experiment for verification of screening results

[0036] The verification process is as follows:

[0037] 1) First, a radiation aging model of BM-MSCs was constructed, and cell viability, apoptosis and senescence were detected at different time points under radiation doses of 3Gy, 6Gy, 9Gy, 12Gy and 15Gy.

[0038] 2) CCK8 experiment was used to study the effect of different radiation doses on the proliferation of BM-MSCs.

[0039] BM-MSCs were divided into 5 experimental groups and 1 control group. The experimental groups were irradiated with 3 Gy, 6 Gy, 9 Gy, 12 Gy and 15 Gy radiation doses, respectively. Figure 3 As shown in the figure, with the increase of radiation dose, the survival rate of BM-MSCs gradually decreased; but when the radiation dose was ≥9Gy, the survival rate of BM-MSCs did not decrease significantly with the increase of radiation dose. This shows that when the radiation dose is ≥9Gy, BM-MSCs have good radiation tolerance. In addition, the survival rate of BM-MSCs on the 7th day did not decrease significantly compared with the survival rate of BM-MSCs on the 4th day, indicating that the effect of radiation on the survival rate of BM-MSCs may reach its peak on the 4th day.

[0040] 3) Using β-galactosidase staining experiments, the senescence of BM-MSCs at 1, 4, and 7 days after irradiation with 3Gy, 6Gy, 9Gy, 12Gy, and 15Gy was investigated. Figure 4 As shown in (a)-(c), the senescent proportion of BM-MSCs increases with the increase of radiation dose and time. For example, on the first day after 9Gy radiation, the senescent proportion of BM-MSCs was 33.6%, which increased to 58.0% on the fourth day and 68.1% on the seventh day. After 12Gy radiation, the senescent proportion of BM-MSCs was 48.1%, 67.1% and 74.3% on the first, fourth and seventh days, respectively; after 15Gy radiation, the senescent proportion of BM-MSCs was 60.9%, 74.5% and 80.3% on the first, fourth and seventh days, respectively.

[0041] 4) Flow cytometry was used to detect the apoptosis rate of BM-MSCs on days 1, 4, and 7 after irradiation with 3Gy, 6Gy, 9Gy, 12Gy, and 15Gy. We found that the apoptosis rate of BM-MSCs did not change significantly on days 1 and 4 under different radiation doses, indicating that BM-MSCs have good radiation tolerance. However, the apoptosis rate of BM-MSCs itself increased significantly on day 7 (see Figure 4 d).

[0042] Therefore, we determined the radiation dose of 9 Gy and 4 days after radiation as the experimental conditions for detecting the effects of small molecule compounds on BM-MSCs senescence.

[0043] 5) Study the effects of 20 small molecule compounds on the activity and senescence of BM-MSCs on the 4th day before and after 9Gy irradiation. BM-MSCs were divided into 21 groups, and each group was added with a screened small molecule compound to detect the survival rate and senescence of the cells. Then, the cells were irradiated with 9Gy ray and the survival rate and senescence of the cells were detected. The results are shown in Figure 2. Figure 5 As shown, compounds 18 and 19 (see the structural formula below) had no significant effect on the activity of BM-MSCs, but after 9Gy irradiation, the activity of BM-MSCs added with compounds 18 and 19 was slightly higher than that of the irradiation control group, and the senescence ratio decreased significantly from 70.1% to 24.2% and 30.4%, respectively. Figure 5 ). This indicates that compounds 18 and 19 have a significant blocking effect on radiation-induced senescence of BM-MSCs. Compounds 5 and 17 also have a good blocking effect on radiation-induced senescence of BM-MSCs, but they have certain toxicity to cells. Therefore, compounds 18 and 19 were selected as target molecules to study their inhibitory effects on radiation damage to BM-MSCs.

[0044]

[0045] In addition, compounds 18 and 19 also have a good inhibitory effect on BM-MSCs apoptosis after radiation. The apoptosis rate of unirradiated BM-MSCs on the 4th day of culture was 4.4%; after adding compounds 18 and 19, the apoptosis rate of BM-MSCs was 4.2%. Therefore, compounds 18 and 19 have almost no effect on BM-MSCs apoptosis. On the 4th day after 9Gy radiation, the apoptosis rate of BM-MSCs was 14.8%; while the apoptosis rates of BM-MSCs added with compounds 18 and 19 were 5.5% and 4.3% respectively on the 4th day after 9Gy radiation (see Figure 6 Compounds 18 and 19 had a good inhibitory effect on radiation-induced BM-MSCs apoptosis.

[0046] Western blot experiments showed that compounds 18 and 19 did not inhibit the expression of p21 protein and CDK2 protein, indicating that compounds 18 and 19 may block radiation-induced senescence of BM-MSCs by inhibiting the interaction between p21 and CDK2 / Cyclin A.

[0047] Conclusion: The screening method of the present invention can effectively screen out interface inhibitors 18 and 19 targeting p21-CDK2 / CyclinA interaction, and the inhibitors show potential application value in inhibiting ionizing radiation-induced cell senescence.

Claims

1. A method for screening a p21-CDK2 / Cycl in interface inhibitor, wherein the inhibitor can inhibit cell senescence, the method comprising the following steps: 1) Construct a model of p21-CDK2 / cyclin A protein interaction; 2) Using the molecular dynamics software AMBER, the binding free energy of the model in step 1) is calculated and the interacting amino acid sites are analyzed by simulation; 3) Using molecular docking software to virtually screen small molecule compound inhibitors based on the simulated protein-protein interaction amino acid regions; optionally, 4) The activity of the screened small molecule compounds was verified using a cell radiation-induced aging model.

2. The method according to claim 1, wherein the cell senescence refers to the senescence of cells after radiation damage.

3. The method according to claim 1, step 1) further comprises using the Alphafold protein structure database and the PDB protein database to construct the p21 and CDK2 / cyclin A protein structures respectively, using ZDOCK software to complete the simulated docking of the p21 and CDK2 / cyclin A protein complex, and screening out the p21-CDK2 / cyclin A protein interaction model that is most similar to the p27-CDK2 / cyclin A protein complex structure.

4. The method according to claim 1, step 2) further comprises using AMBER software to perform molecular dynamics simulation, data analysis, binding free energy calculation, and hot spot amino acid residue simulation analysis on the protein model obtained in step 1) to obtain key amino acid residue sites involved in protein interaction.

5. The method according to claim 4, wherein the key amino acid residue sites are Gln254 and Thr282 amino acid residues of Cyclin A.

6. The method according to claim 1, step 2) further comprises using Autodock_Vina molecular docking software to virtually screen out small molecule compound inhibitors that inhibit protein-protein interactions based on key amino acid residue sites of protein interactions.

7. The method according to claim 1, wherein the verification in step 4) comprises: a) Using gamma rays to irradiate bone marrow mesenchymal stem cells (BM-MSCs) to construct a cell radiation-induced aging model; b) After the small molecule compound screened in step 3 is co-incubated with BM-MSC, the cells are irradiated with gamma rays, and the inhibitory activity of the small molecule compound on cell radiation-induced senescence is detected by β-galactosidase staining experiment and / or flow cytometry.

8. Use of the compound of the following formula or its pharmaceutically acceptable salt in the manufacture of an anti-cell aging drug:

9. The use according to claim 8, wherein the cell senescence refers to radiation-induced senescence.

Images